Chemistry Reference
In-Depth Information
The variation of the normalized STD responses for different protons within a ligand also
contains information on
how
the ligand binds to the target protein.
[
96, 97
]
The magnitude
of a normalized STD response is related to the corresponding ligand proton proximity to
target protons, a consequence of the distance dependence of intermolecular
1
H-
1
H cross-
relaxation. Thus, a stronger STD response implies closer intermolecular contact, which
can be interpreted as information on which part of the ligand is responsible for most of the
binding energy. To compare directly the normalized STD responses, the same assumptions
as described above should be fulfilled. This 'ligand epitope mapping' approach is best
suited for weakly binding ligands and/or the use of sufficiently short saturation times, in
order to ascertain that the magnetizations of the ligand protons are not equalized due to
spin diffusion during the lifetime of the ligand-target complex. This approach has been
substantially extended to determine indirectly the conformation of a ligand when bound
to a target protein with a known structure. In this method, called SOS-NMR,
[
98
]
STD is
applied to a ligand complexed to a series of perdeuterated target protein samples, each with
different specific amino acid types protonated. The relative STD intensities of the ligand
peaks from the different samples contain quantitative information on which protons on the
ligand are in the vicinity of which type of amino acid in the target protein. With a known
target protein structure, the structure of the ligand-target complex can be deduced from
this information provided that experiments have been performed on a sufficient number
of differently labeled protein samples to define the binding site uniquely. An analysis of
272 unique crystal structures of ligand-protein complexes showed that 3-9 differently
labeled protein samples would be enough to identify unambiguously the ligand binding
site of more than 90% of the ligand-protein complexes. Obviously, SOS-NMR is a very
resource-intensive method, but could prove highly valuable in cases where it has not been
possible to obtain any structural information on the ligand-target complex either by X-ray
crystallography or by 'traditional' NMR methods.
Finally, STD has been demonstrated to be applicable for the detection of ligand binding
in very demanding systems such as a virus
[
99
]
and an integral membrane protein either
reconstituted in liposomes
[
100
]
or on living cells.
[
101
]
WaterLOGSY.
TheWaterLOGSYexperiment
[
73, 74
]
relies on water-mediated magnetization
transfer to compounds that bind to the target protein. The most favored version of this
experiment
[
102
]
is essentially an NOE experiment starting with selective inversion of the
bulk water magnetization followed by a long mixing time (up to several seconds). The
inverted bulkwatermagnetization is transferred to the target protein and binding compounds
via several possible magnetization transfer pathways:
[
6
]
(i) direct
1
H-
1
H cross-relaxation
between compounds and tightly bound water molecules at the binding site, (ii) chemical
exchange of inverted bulk water with protein hydroxyl and amine groups at the binding
site, which in turn will transfer the inverted magnetization to the bound compound protons,
and (iii) chemically exchanging hydroxyl and amine groups on the protein that are not
situated in the binding site may contribute via spin diffusion through the protein. During
the mixing time, the
1
H-
1
H cross-relaxation will give rise to NOEs with opposite signs
depending on whether the magnetization transfer takes place in the bound state (slow
tumbling, long rotational correlation time) or in solution (fast tumbling, short rotational
correlation time). Reversibly binding compounds will experience magnetization transfer
in both the bound and free states, whereas nonbinding compounds will only experience
